G-protein coupled receptors (GPCRs) appear promising as targets for promoting osteogenesis. Agonists of several GPCR systems are potent inducers of new bone formation. Similar to GPCR systems in other tissues, the effects of GPCRs in bone are likely to be regulated. This regulation may involve post-translational modifications of key components of the signaling cascade and/or interactions with regulatory molecules. Of these mechanisms, the most extensively studied is direct phosphorylation of receptor proteins. Phosphorylation of GPCRs is responsible for the rapid attenuation of GPCR signaling that occurs in the continuous presence of agonist. This desensitization may limit the beneficial effects of GPCR agonists on bone. Understanding the mechanisms that that regulate GPCR function might therefore lead to novel strategies for promoting new bone formation. Accumulating evidence suggests that agonist-specific desensitization of GPCRs is largely mediated by direct phosphorylation of receptor serine and threonine residues by a recently discovered family of receptor specific kinases (GPCR kinases or GRKs). Following agonist stimulation, GRKs directly phosphorylate GPCRs and attenuate GPCR signaling. Of the GPCR agonists that stimulate osteogenesis, parathyroid hormone (PTH) has been the most extensively studied. When given to either animals or humans, PTH is anabolic for bone. In the proposed studies, we hypothesize that GRKs play important roles in modulating GPCR function in osteoblasts and propose to investigate the role of these kinases in osteogenesis using the PTH receptor as a model system. To test this hypothesis, we will use three complementary approaches. First, we will determine the structural components of PTH receptor required for GRK actions using cells that have been transfected with the PTH receptor cDNA. This approach will permit mutagenesis studies, as well as isolation of epitope-tagged PTH receptors by immunoprecipitation and direct examination of their phosphorylation state. Second, we will study the role of GRKs in modulating PTH receptor signaling in osteoblast cell lines. These studies will evaluate the patterns of GRK expression in osteoblasts, as well as the effect of modulating GRK activity on PTH receptor responsiveness, extracellular matrix accumulation, apoptosis and production of osteoblastic factors which modulate osteoclastogenesis. Lastly, we will create transgenic mice that overexpress either GRKs, or GRK inhibitory peptides in a bone-specific manner. After characterizing their bone phenotype, these transgenic animals will be used to investigate the effect of different PTH dosing regimens on bone formation. These studies should provide new, important insights into the role of GRKs in regulating osteogenesis. Understanding the biochemical mechanisms that regulate hone formation could lead to the development of novel strategies for treating a wide variety of bone disorders that cause osteoporosis.